Expanding the Spectrum of BRAF Non-V600E Mutations in Thyroid Nodules: Evidence-Based Data from a Tertiary Referral Centre

The BRAF p.V600E mutation represents the most specific marker for papillary thyroid carcinoma and is potentially related to aggressive behavior and persistent disease. BRAF alterations other than the p.V600E are less common in thyroid carcinoma and represent an alternative mechanism of BRAF activation with unclear clinical significance. The study aims to describe the frequency and clinicopathologic characteristics of BRAF non-V600E mutations in a large cohort (1654 samples) of thyroid lesions characterized by next-generation sequencing. BRAF mutations have been found in 20.3% (337/1654) of thyroid nodules, including classic (p.V600E) mutation in 19.2% (317/1654) of samples and non-V600E variants in 1.1% of cases (19/1654). BRAF non-V600E alterations include 5 cases harboring p.K601E, 2 harboring p.V600K substitutions, 2 with a p.K601G variant, and 10 cases with other BRAF non-V600E alterations. BRAF non-V600E mutations have been reported in one case of follicular adenoma, three cases of conventional papillary thyroid carcinoma, eight cases of follicular variant of papillary carcinomas, one case of columnar cell variant papillary thyroid carcinoma, one case of oncocytic follicular carcinoma, and two bone metastasis of follicular thyroid carcinoma. We confirm that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential. However, in both aggressive cases, the BRAF mutations were concomitant with other molecular alterations, such as TERT promoter mutation.


Introduction
Diagnostic thyroidectomy is currently the recommended definitively procedure to exclude malignancy in patients with worrisome thyroid nodules. Molecular testing is commonly used to characterize tumors of follicular cell derivation: the BRAF p.V600E, RAS point mutations, and fusion oncogenes (e.g., RET/PTC fusion gene) are the most frequent molecular alterations [1][2][3][4][5]. BRAF gene codes for a protein belonging to a family of serine-threonine protein kinases that also includes ARAF and CRAF. RAF proteins play an important role in the MAP kinase signaling cascade, one of the pathways involved in cell proliferation and differentiation.
BRAF mutations are found in 60-80% of papillary thyroid carcinomas (PTC), and the most frequent alteration is the BRAF p.V600E. This mutation consists of a single nucleotide substitution involving the thymine in position 1799 (transversion, c.1799T > A). This mutation results in a valine to glutamate substitution at amino acid 600 (BRAF p.V600E) that mimics BRAF phosphorylation, causing constitutive activation of the protein. This activation leads to increased and uncontrolled cell proliferation necessary for tumor transformation and promotion. A previous study has demonstrated that BRAF mutation may be heterogeneously distributed in PTCs [6]. In PTCs, the BRAF p.V600E mutation has been associated with extrathyroidal extension, lymph nodal and distant metastases, higher stage, and risk of recurrence.
The aim of the present study was to report the experience of the Bologna molecular pathology laboratory regarding the frequency of BRAF non-V600E alterations in a large clinical cohort of thyroid nodules.
Follicular variant papillary thyroid carcinoma (FV-PTC) was the most common histological subtype harboring BRAF non-V600E mutations (8 of 19 cases, 42.1%). BRAF non-V600E mutations were also observed in one case of follicular adenoma (5.3%), three cases of conventional papillary thyroid carcinoma (15.8%), one case of columnar cell variant papillary thyroid carcinoma (5.3%), one case of oncocytic carcinoma (5.3%), and two bone metastases of follicular thyroid carcinoma (10.5%). Tumor capsule was present in five cases (Tables 3 and 4). An extrathyroidal extension was observed in one classic papillary carcinoma (CL-PTC) (case #19- Table 4), while vascular invasion was detectable in three cases (Tables 3 and 4). Cases with BRAF non-V600E mutations were mostly pT1 (10 of 13, 76.9%), including seven pT1a and three pT1b (Table 3). According to the AJCC (American Joint Committee on Cancer) prognostic stage, eleven cases were stage I, and one was stage II (Tables 3 and 4). The vast majority of the samples harboring non-V600E were indolent thyroid lesions, but in two cases, distant (bone) metastases were detected at presentation (case #2, #3-Tables 2 and 4, Figure 1).

Discussion
BRAF encodes the BRAF protein, which belongs to a family of serine-threonine protein kinases, including ARAF and CRAF. RAF proteins play an important role in the MAP kinase signaling cascade, one of the main pathways that regulate cell proliferation and differentiation. Mutant BRAF has been implicated in the pathogenesis of different types of cancer, including thyroid carcinoma, colorectal carcinoma, non-small cell lung carcinoma, ovarian carcinoma, gliomas, melanoma, and gastrointestinal stromal tumor (GIST) [14].
In this study, we investigated the prevalence and clinical significance of BRAF non-V600E variants in a large cohort of cases from a tertiary referral center. Next-generation sequencing allowed the detection of BRAF non-V600E mutations in 1.2% of 1654 thyroid nodules. BRAF non-V600E mutations are uncommon. They are typically found in indolent follicular-patterned tumors, and thus-in spite of being BRAF mutated forms-belong to the spectrum of thyroid tumors with RAS-like features (instead of belonging to the spectrum of BRAF-V600E-like tumors). In the TCGA cohort, of 240 mutated cases, 5 (2.1%) harbored non-V600E mutations. All of them were FV-PTC with p.K601E substitution or inframe deletions/insertion (cBioPortal-"Thyroid Carcinoma-TCGA", Firehose Legacy).
BRAF p.K601E mutation represented the most common BRAF non-V600E alteration and, in agreement with other published case series, was only detected in follicular-patterned tumors (see Table 4) [15,16]. BRAF p.K601E has been reported in a few cases of follicular thyroid carcinoma [11,17,18], as well as in one case of NIFTP [19] and four cases of follicular adenoma [11,[20][21][22]. Similar to previous reports, we found BRAF p.K601E in follicular variant PTC (four cases) as well as in one case of follicular adenoma. In our series, K601Emutant FV-PTC showed no aggressive features, such as extrathyroidal extension, vascular invasion, or metastases, supporting the notion that BRAF p.K601E mutated thyroid nodules are typically indolent, in contrast to BRAF p.V600E mutated carcinomas [23,24]. In addition to BRAF p.K601E, we identified other non-V600E mutations not only in follicular-patterned tumors (two FV-PTC and three FTC) but also in three classical PTCs and in one CCV-PTC. Interestingly, a recent study reported that CCV-PTC appears to harbor a higher incidence of non-V600E variants [25].
Among BRAF non-V600E and non-K601E mutations, BRAF p.T599del was detected in one FV-PTC. This mutation has previously been reported in an adenomatous goiter [18,26]. BRAF p.K601_W604del was found in one of our FV-PTC cases. The mutation has previously been reported in papillary thyroid carcinoma [26,27]. BRAF p.T599_V600insEAT, detected in one classical PTC from our cohort, has never been previously reported in thyroid lesions but was identified in a single case of Langerhans cell histiocytosis [26,28]. We found one BRAF exon 11 mutation (p.G469S) in a case of FV-PTC. This mutation, too, had never been previously reported in thyroid lesions, but it was identified in hematopoietic and in solid tumors (e.g., melanomas, lung adenocarcinoma, and bile duct adenocarcinoma) [26].
The vast majority of the samples harboring BRAF non-V600E mutations were indolent thyroid lesions. However, we identified BRAF non-V600E mutations in two FTC patients with distant (bone) metastases at presentation. One FTC harbored BRAF p.A598_T599insILA and the other BRAF p.K601N. BRAF p.A598_T599insILA has never been previously reported. BRAF p.K601N has been reported in rare cases of FTC, thyroid lymphoma, lung carcinoma, colorectal carcinomas, and melanoma [26,29,30]. Intriguingly, the two cases with aggressive behavior harbored another mutation other than BRAF non-V600E: in one case, a pathogenic TERT promoter mutation was detected (case #2, Table 2); the other case harbored a 19q imbalance (case #3, Table 2) [30].
In conclusion, our results expand the spectrum of BRAF non-V600E mutations in thyroid nodules. We have confirmed that BRAF non-V600E mutations are uncommon and typically found in indolent follicular-patterned tumors. We report for the first time three BRAF non-V600E variants previously undetected in thyroid tumors, including one concomitant with TERT mutation and associated with distant metastasis. Indeed, we show that BRAF non-V600E mutations can be found in tumors with metastatic potential if associated with other molecular alterations. Thus, BRAF non-V600E mutated tumors should be thoroughly investigated, looking for thyroid tumor progression-related molecular changes such as TERT promoter mutation.

Case Selection
Data about 1654 thyroid nodules subjected to BRAF mutation analysis from January 2011 until June 2019 were collected from the database of the solid tumor molecular athology laboratory (Bologna, Italy). Analyses were performed starting from fine-needle aspiration specimens ("Direct fine needle aspiration cytology specimen" or "Stained cytological smear") or surgical material (formalin-fixed and paraffin-embedded tissue-FFPE). The cytopathological and histopathological review was performed by two expert pathologists (GT, ADL) according to World Health Organization classification of thyroid malignancy [31].

DNA Extraction and BRAF Analysis
DNA from FFPE specimens was extracted starting from 2 to 4 serial 10 µm-thick sections using the QuickExtract FFPE DNA extraction kit (LGC Biosearch Technologies, Berlin, Germany), scraping the area of interest, according to the selection performed by a pathologist on final hematoxylin and eosin (E&H) slide. DNA was quantified using Qubit dsDNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA from cytological specimens was extracted using the MasterPure DNA purification kit (LGC Biosearch Technologies, Berlin, Germany), according to the manufacturer's instructions.  Institutional Review Board Statement: All the experimental procedures were carried out in accordance with the general authorization to process personal data for scientific research purposes from "The Italian Data Protection Authority" (http://www.garanteprivacy.it/web/guest/home/docweb/ -/docwebdisplay/export/2485392 (accessed on 29 January 2021)). All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Helsinki Declaration (https://www.wma.net/fr/news-post/en-matierede-transfertdes-taches-la-securite-des-patients-et-la-qualitedes-soins-devraient-etre-primordiales/ (accessed on 29 January 2021). The study did not affect the clinical management of the involved patients' samples.

Informed Consent Statement:
Written informed consent for mutational analysis was obtained from all patients. Information regarding the human material was managed using anonymous codes, and all samples were handled in compliance with the Helsinki Declaration.